Type 2 diabetes mellitus (DM2) and its association with metabolic derangements such as obesity represent an enormous public health burden for which additional therapies are desperately needed (34). Historically, the metabolic derangements characteristic of DM2 were attributed to defects in insulin action on target tissues such as muscle, liver, and fat. Accumulating evidence now implicates the central nervous system (CNS) as a key insulin target organ in the modulation of energy balance. Defects in CNS insulin action result in hyperphagia, weight gain, and dysregulated hepatic glucose production (42). Insulin action in any tissue is dependent on successful transport of insulin from the bloodstream across the endothelium into the target organ; in skeletal muscle this transendothelial transport functions as the critical rate limiting step in insulin action (9). It nw appears that insulin traverses the endothelial cell of skeletal muscle via interactions between the insulin receptor (IR) and caveolae (55), plasma membrane invaginations import for signaling and transport (40). Furthermore this transport is dependent on functional signaling through the IR (56). Historical data suggests that insulin entry into the brain occurs via IR-mediated transendothelial transport (5, 14, 48). However the exact mechanism insulin uses to cross the endothelial cells of the CNS, which form a different endothelial-tissue barrier than those in skeletal muscle (59), remains unexamined. The following strategies will be used to test the hypothesis that insulin transendothelial transport in the CNS occurs via caveolae-mediated process dependent on functional insulin signaling. First, physical interactions between the IR and caveolin-1, the key component protein of caveolae, will be examined using protein-protein interaction studies as well as microscopy based colocalization studies in whole brain, isolated brain microvessels, and primary rat brain endothelial cells. Rat brain endothelial cell lines as well as primary rat brain endothelial cells will then be employed to determine if insulin uptake into endothelial cells is affected by inhibitors of caveolae formation or inhibitors of insulin signaling. Completion of these experiments will increase understanding of CNS insulin transport mechanisms. These mechanisms can then be further explored in future experiments in animal models of diabetes and obesity. As CNS insulin action is critical for controlling weight and energy balance, these transport mechanisms will likely represent attractive future pharmacologic targets in the treatment of obesity and DM2. PUBLIC HEALTH RELEVANCE: Type 2 diabetes represents a significant public health concern and abnormalities in insulin action within the brain likely contribute to its pathogenesis. This project attempts to increase understanding of insulin transport into the brain in order to provide future targets for pharmaceutical development in the treatment of type 2 diabetes.